Respiratory syncytial virus ribonucleoproteins hijack microtubule Rab11 dependent transport for intracellular trafficking

Respiratory syncytial virus (RSV) is the primary cause of severe respiratory infection in infants worldwide. Replication of RSV genomic RNA occurs in cytoplasmic inclusions generating viral ribonucleoprotein complexes (vRNPs). vRNPs then reach assembly and budding sites at the plasma membrane. However, mechanisms ensuring vRNPs transportation are unknown. We generated a recombinant RSV harboring fluorescent RNPs allowing us to visualize moving vRNPs in living infected cells and developed an automated imaging pipeline to characterize the movements of vRNPs at a high throughput. Automatic tracking of vRNPs revealed that around 10% of the RNPs exhibit fast and directed motion compatible with transport along the microtubules. Visualization of vRNPs moving along labeled microtubules and restriction of their movements by microtubule depolymerization further support microtubules involvement in vRNPs trafficking. Approximately 30% of vRNPs colocalize with Rab11a protein, a marker of the endosome recycling (ER) pathway and we observed vRNPs and Rab11-labeled vesicles moving together. Transient inhibition of Rab11a expression significantly reduces vRNPs movements demonstrating Rab11 involvement in RNPs trafficking. Finally, Rab11a is specifically immunoprecipitated with vRNPs in infected cells suggesting an interaction between Rab11 and the vRNPs. Altogether, our results strongly suggest that RSV RNPs move on microtubules by hijacking the ER pathway.


Abstract:
The respiratory syncytial virus (RSV) is the primary cause of severe respiratory infection in infants worldwide. Replication of RSV genomic RNA occurs in cytoplasmic inclusions generating viral ribonucleoprotein complexes (vRNPs). vRNPs then reach assembly and budding sites at the plasma membrane. However, mechanisms ensuring vRNPs transportation are unknown. We generated a recombinant RSV harboring fluorescent RNPs allowing us to visualize moving vRNPs in living infected cells and developed an automated imaging pipeline to characterize the movements of vRNPs at a high throughput. Automatic tracking of vRNPs revealed that around 10% of the RNPs exhibit fast and directed motion compatible with transport along the microtubules. Visualization of vRNPs moving along labeled microtubules and restriction of their movements by microtubule depolymerization further support microtubules involvement in vRNPs trafficking. Approximately 30% of vRNPs colocalize with Rab11a protein, a marker of the endosome recycling (ER) pathway and we observed vRNPs and Rab11labeled vesicles moving together. Transient inhibition of Rab11a expression significantly reduces vRNPs movements demonstrating Rab11 involvement in RNPs trafficking. Finally, Rab11a is specifically immunoprecipitated with vRNPs in infected cells suggesting an interaction between Rab11 and the vRNPs. Altogether, our results strongly suggest that RSV RNPs move on microtubules by hijacking the ER pathway. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Unfunded studies Enter: The author(s) received no specific funding for this work.  worldwide and is increasingly recognized as a major respiratory pathogen in the elderly and frail. Yet, 35 no curative treatment or vaccine is currently marketed. The late stages of RSV multiplication remain 36 poorly understood despite their potential targets for the development of antiviral strategies. In the 37 infected cell, the viral genome is encapsidated and associated to the viral polymerase, to form the viral 38 ribonucleoprotein (vRNP). The vRNPs are produced and assembled in cytoplasmic viral factories. The 39 process ensuring their transport to the budding sites, at the plasma membrane, awaits to be precisely 40 defined. Here we explored these mechanisms by tracking moving vRNPs in living infected cells. We 41 developed an automated imaging pipeline allowing us to characterize the movements of vRNPs, at an 42 unprecedented throughput. We then exploited the potential of our method to monitor the behaviour 43 of the vRNPs during the infection. Using this approach, we document substantial trafficking of RSV 44 along the microtubule network and demonstrate that RSV hijacks the recycling endosome pathway to 45 promote the mobility of its vRNPs. Altogether, this work provides a cutting-edge approach allowing for 46 Introduction 50 Respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in 51 children worldwide. RSV infections are responsive for around 120 000 child death per year mostly in 52 developing countries and are the main cause of child's hospitalization in western countries (1,2). 53 Periodic reinfections occur throughout life. Considered as benign in healthy children and adults, RSV 54 infections are increasingly associated with significant morbidity and mortality in elderly and 55 immunocompromised people with much the same disease burden as for influenza (3)(4)(5). Despite the 56 high burden of RSV infection, there are still no vaccine nor curative treatment available. Search for 57 antiviral drugs is active, most of the candidates in development targeting entry steps and viral RNA 58 synthesis (6). 59 RSV belongs to the Mononegavirales order. Its 15 kb negative single stranded genomic RNA encode 11 60 proteins. RSV is an enveloped virus exhibiting two major surface glycoproteins G and F. The matrix 61 protein M coats the inner side of the viral membrane and surrounds the viral genomic material (7). 62 The genomic RNA is tightly encapsidated by the nucleoprotein N and forms an helicoidal 63 ribonucleocapsid. N is further bound to the polymerase (L) and its viral cofactors, the phosphoprotein 64 P and M2-1, to form the viral ribonucleoprotein (vRNP) (8-10). RNPs are the functional units driving 65 viral RNA synthesis. Using the ribonucleocapsid as a template, L and P proteins ensure both the viral 66 transcription and replication processes, while M2-1 is selectively required for the transcription process. 67 Viral RNA synthesis occurs within cytoplasmic inclusions, referred to as inclusion bodies (IBs), which 68 can be regarded as viral factories (11). The newly synthetized RNPs then reach the plasma membrane, 69 where RSV presumably buds, forming elongated membrane filaments (12)(13)(14). The matrix protein M 70 is thought to drive virion assembly by bridging RNPs with the plasma membrane through its 71 interactions with the RNPs and the cytoplasmic tail of the viral F glycoprotein (14-19). The exact 72 location of final virus assembly is debated since recent analysis of live G trafficking showed that RNP 73 assemble with membrane glycoprotein prior insertion to plasma membrane (20). In any case, 74 trafficking of RNPs to the cell surface requires active transport mechanisms, since diffusion of large 75 objects in the cytoplasm is restricted by molecular crowding by organelles, the cytoskeleton and high 76 protein concentrations (21). This active transport might involve hijacking of the actin networks (22,23) 77 or of the recycling endosome (RE) pathway. RE is involved in delivering endocytosed material as well 78 as cargos from the trans Golgi network to the plasma membrane (24). Interestingly RE pathway has 79 been implicated in RNP export of numerous viruses such as the influenza, Sendai, measles and mumps 80 viruses (25-28). The RE pathway is regulated by the small GTPase Rab11 which is present in three 81 isoforms (Rab11a, Rab11b and Rab25) in human cells. The GTP bound active form of Rab11 binds to 82 target vesicle' membrane thanks to its GPI anker and recruits specific factors ensuring transportation, 83 docking or fusion of the vesicle to its cognate membrane (29). Rab11 vesicle can traffic along both 84 microtubules and actin networks by engaging various molecular motors by means of molecular 85 adaptors called Rab11 Family Interacting Proteins (Rab11-FIPs). 86 Here we engineered recombinant RSV expressing a fluorescent N to visualize the moving RNPs in living 87 infected cells. An automatic quantitative analysis of RNPs trajectories reveals rapid and directional 88 motions that were abolished by nocodazole treatment consistent with transportation along the 89 microtubule network. We observed a colocalization of around one third of the RNPs with Rab11a and 90 RNPs moving together with Rab11a in infected cells. Inhibition of Rab11a expression impairs RNPs 91 movements. Interestingly RSV infection affects transferrin recycling, dependent of Rab11 pathway. 92 Moreover, interaction between Rab11a and viral RNPs was evidenced by co-immunoprecipitation 93 experiments. All of these data confirm that RSV is high jacking the Rab11-RE pathway to ensure RNPs 94 export. 95 96

Development of recombinant RSV to monitor intracellular transport of RNPs. 98
To investigate RNPs dynamics in living cells, the RSV N protein was fused to a fluorescent tag to 99 generate fluorescent RNPs. We previously demonstrated that a N-ter fusion of the N doesn't affect the 100 transcription-replication activities if co-expressed with a wild type N (11). We thus engineered and 101 rescued a recombinant RSV expressing both the wild type N and a GFP-N by inserting the GFP-N coding 102 sequence flanked with transcription start and stop signals sequences between M and SH genes in our 103 reverse genetic vector ( Fig S1a) (30). To ascertain whether the GFP-N would interfere with viral growth, 104 we compared single cycle growth kinetics of wild type and N-GFP expressing RSV (hereinafter referred 105 to as RSV-GFP-N) ( Fig S1b). The RSV-GFP-N growth was similar to the wild type RSV and reached titers 106 around 10 6 PFU/mL in 24h, showing that the GFP-N addition did not significantly impair viral 107 multiplication. We then investigated GFP-N protein localization in respect to the wild type N protein 108 and known viral structures such as IBs and viral filaments. HEp-2 cells were infected by RSV-GFP-N for 109 24h and intracellular localization of RSV N, P and F proteins were determined by confocal imaging after 110 immunostaining of each protein. The GFP signals were recorded in parallel. As previously described, 111 IBs, cytoplasmic dots and viral filaments were observed by immunostaining of N and P, and F staining 112 only revealed viral filaments (Fig 1) (31). The GFP signals perfectly colocalize with N and P staining in 113 IBs, viral filaments and small puncta in the cytoplasm. Of particular interest, we noted a perfect 114 colocalization of GFP signals with both N and P staining in cytoplasmic dots which could be considered 115 as RNPs (Fig 1 and S1c). Moreover, we observed colocalization of the GFP signals with F staining in viral 116 filaments (Fig 1c, panel F). These data strongly suggest that GFP-N is incorporated together with the 117 wild type N in the vRNPs. Altogether, the RSV-GFP-N was considered suitable as a novel tool marker to 118 assess vRNPs trafficking across the cytoplasm during infection. 119 slight changes of fluorescence within IBs were prone to induce false-positive signals (Fig S2a). We also 137 filtered tracks, in which particles instant speeds always remained below 50% of their maximal instant 138 speed along the entire track recorded to remove artifactual links between the tracks of two slow 139 moving objects (See Fig S2b and legend). 140 We then quantified several parameters characterizing intracellular movement of vRNPs. Displacement 141 is characterized by 1) the track length which corresponds to the sum of lengths of displacement 142 between two time points and 2) the track displacement, which corresponds to the minimum distance 143 between the first and the last position of the particle (Fig 2b). Track velocity is the ratio between track 144 displacement and track duration. Track displacement and track velocity are thus expected to be high 145 for particles exhibiting long range-oriented motion. Smoothed instant speed is the ratio between the 146 minimum distance between the first and the fourth of 4 consecutive positions and the time interval 147 between these positions (see Fig S2f and corresponding legend). Focusing on instant velocities of single 148 fast-moving particles, we observed that rapid motions occur intermittently (Fig 2c). Thus, we also 149 calculated the track maximum speed corresponding to the maximum smooth instant speed of the 150 track. Cumulative distribution of track displacement for 6 cells in one experiment is shown in Figure  151 2e. Around 10% of the tracked particles exhibit a displacement over 1.8 µm when most of them exhibit 152 small displacement (median ranging from 0.29 to 0.42µm). Likewise, cumulative analysis of particles 153 maximal speeds shows that around 10% of the RNPs exhibit maximum velocities above 1.7µm/s (Fig  154   2d). The median value of each parameter reflects the overall characteristics of the particle's 155 movements in an individual cell and provides a single value that can be used for further comparing 156 treated groups. Median of track maximal speeds, track velocities and track displacements are not 157 significantly different in 4 independent experiments on two different cell types (HEp-2 or A549). This 158 points to a potential generalizability of vRNP's behavior during RSV infection in this model and 159 indicates that these parameters are suitable for further group comparisons (Fig 2f, 2g, 2h).

Rab11 and RNPs colocalize in infected cells. 224
Recycling endosome vesicles, marked by the small GTPase Rab11, are known to be involved in RNPs 225 trafficking of several negative strand RNA viruses (25,26,28,34). To investigate the recycling endosomes 226 involvement in RSV RNPs trafficking, we analyzed Rab11a and RSV RNPs intracellular localizations. We 227 infected A549 cells constitutively expressing HA-Rab11a (A549-HA-Rab11a) with wild type RSV (35). At 228 the indicated time points, cells were fixed and N and HA-Rab11a were detected by immunostaining. 229 Both N and HA staining revealed numerous individual cytoplasmic dots corresponding respectively to 230 vRNPs and Rab11a positive endosomes, which exhibited partial colocalization (Fig 5). To quantify the 231 colocalisation between Rab11a and N positives dots, we extracted spot detections and performed 232 object-based colocalization analysis using Icy software. This method first segments the spots and then 233 analyzes their relative distribution with second order statistics enabling robust quantification of 234 colocalized objects (36). This analysis revealed that approximately 30% of N spots colocalize with 235 Rab11a positive vesicles at 12 to 18h p.i. with a slight decrease at 24h p.i.. In contrast, less than 10% 236 of N spots colocalize with EEA1, a marker of early endosomes used as a negative control. Similar results 237 were obtained in A549 and HEp-2 cells (Fig S4). These data suggest an involvement of Rab11a in vRNPs 238 traffic. 239 240 241 242

Rab11 pathway is involved in RNPs fast and directed movements 252
To determine if Rab11a could be involved in RNPs trafficking, we set up an experiment to visualize both 253 RNP and Rab11 movements in living infected cells. A549 and HEp-2 cells were infected with RSV-GFP-254 N and transiently transfected with a mCherry-Rab11a expression vector. At 18-20h.p.i., we performed 255 dual color time-lapse imaging to assess the dynamic of both Rab11a and RNPs. Interestingly, we 256 observed fast-moving RNPs moving together with Rab11a positive vesicles demonstrating that at least 257 some of the fast-moving RNPs are associated with Rab11a positive structures (Fig 6a and  258 Supplementary Movie 8). Strikingly, RNPs appeared as pulled up by the Rab11 positive spots. RNPs 259 associated with Rab11 positive vesicles were no longer mobile when cells were treated with 260 nocodazole suggesting that these movements were microtubule-dependent (Supplementary Movie 9). 261 The dual color acquisition settings unfortunately prevent automatic tracking of RNPs and Rab11a in 262 this experiment. 263 To further demonstrate the essential role of Rab11 in RNPs export, we performed SiRNA depletion 264 experiments. A549 cells were transiently transfected with small interfering RNA (SiRNA) targeting 265 Rab11a or control scrambled SiRNA for 48h and subsequently infected with RSV-GFP-N for 18-20h 266 before live imaging. Western blot analysis of the whole cell lysates using an antibody against Rab11a 267 showed a considerable decrease in Rab11a levels in SiRNA targeting Rab11a treated cells compared to 268 the control (Fig. S4c). RNPs movements were quantified as described above. The track velocity, track 269 maximum velocity and track displacement were significantly reduced in Rab11a silenced cells as 270 compared to the control (Fig 6, Supplementary Movies 10 and 11). The centered projection of the 271 tracks from one cell illustrates the reduction of track displacement. These results suggest that Rab11a 272 is involved in RNPs trafficking. 273 Since our data indicate that RSV hijacks the Rab11a pathway for RNP export, we examined whether 274 RSV infection impacted in turn ER function. To this end, we explored transferrin recycling, which is 275 dependent on ER functioning. Mock or RSV infected A549 cells were incubated in the presence of 276 fluorescent transferrin and were then placed in fresh medium. Cytoplasmic fluorescence 277 (corresponding to non-recycled transferrin) was quantified after 0 and 20 min of incubation in fresh 278 medium ( Fig S5). Interestingly, the infected cells exhibit an increase in transferrin recycling as 279 compared to uninfected ones suggesting an acceleration of the traffic of RE. Altogether, these data 280 suggest that RSV not only uses the recycling endosome pathway but also manipulates its functioning.

Biochemical validation of RNP-Rab11a association 295
We further assessed the association between RNPs and Rab11a performing co-immunoprecipitation 296 (IP) analysis. A549 cells stably expressing HA-Rab11a were infected for 16h with RSV-GFP-N or RSV 297 expressing GFP (RSV-GFP) as a control. Cell lysates were incubated with beads coated with anti-GFP 298 antibodies and IP was carried on. Both pre-purification and IP samples were analyzed by western blot 299 to reveal N, P and HA-Rab11 (Fig 7). Wild type N and P proteins were efficiently purified together with 300 the GFP-N protein suggesting that the whole RNP is co-immunoprecipitated with GFP-N. The HA-301 Rab11a protein was also repeatedly co-immunoprecipitated with the GFP-N when no corresponding 302 band was visible in the control. Consistent with the colocalisation and trafficking experiments, only a 303 small fraction of Rab11a was co-immunoprecipitated with the RNPs. The mirror IP was performed on 304 A549-HA-Rab11 cells infected with wild type RSV for 16h using anti-HA antibodies. N protein was found 305 in the IP fraction of Rab11HA expressing cells but not of wild type A549 cells used as control. 306 Interestingly P was also co-immunoprecipitated with HA-Rab11a suggesting that Rab11 is interacting 307 with the RNP. To further investigate this point, we performed similar IP experiments on cells infected 308 with a recombinant RSV expressing a L protein fused to GFP (RSV-L-GFP). The GFP is inserted in the 309 hinge 2 region of L (11). Western blot analysis of the IP fraction failed to reveal the GFP-L most likely 310 due to a lack of sensitivity and poor transfer of a large protein like L. However, N and P were co-311 immunoprecipitated thus validating the IP and confirming the capture of the RNP together with the 312 GFP-L. Finally, we detected HA-Rab11a repeatedly and specifically in the IP sample consistent with an 313 interaction between Rab11a and the RNP.

325
The understanding of many aspects of RSV multiplication has progressed considerably in recent years. 326 However, the late stages of the viral cycle remain poorly understood. In particular, the mechanisms of 327 transport of RSV RNPs from cytoplasmic viral factories to viral assembly sites at or near the plasma 328 membrane remain unexplored. In this study we were able to observe for the first time the movement 329 of RNPs in infected cells using a recombinant RSV. Our strategy was to engineer a recombinant virus 330 expressing an N protein fused to a fluorescent tag to produce fluorescent RNPs. To our knowledge this 331 is the first description of a RSV expressing a tagged N. Expression of this additional GFP-N does not 332 deeply alter the replicative capacities of the virus. Noteworthy, fluorescent N perfectly colocalizes with 333 the wild type N in the viral structures (IBs, RNPs and viral filaments) strongly suggesting that GFP-N is 334 indeed incorporated in RSV RNPs. These data validate the use of the RSV-GFP-N as a new tool to study 335 trafficking of RSV RNPs. In theory, more than 2000 N monomers are required to encapsidate the RSV 336 15kb genomic RNA (10). Due to the transcription gradient, GFP-N is expected to be less abundant than 337 the wild-type N. Nonetheless, the oligomerization of N and GFP-N on the viral RNA leads to an 338 amplification of the fluorescent signal and allows for the detection of vRNPs. The GFP-N virus thus 339 represents a tool of choice for the monitoring of vRNPs in living cells. An alternative strategy based on 340 the labeling of the viral genome with nucleotide probes has been used to analyze the dynamics of RNPs 341 during entry or the movement of viral filaments at the cell surface. However, this method did not 342 enable RNPs trafficking analysis (12,13). vRNPs are small, highly mobile objects. To track them, we had 343 to find a compromise between the number of optical slices (z) analyzed and the time interval between 344 2 images. Our optimized pipeline relies on the acquisition of images on 2 to 3 optical planes with a 345 total thickness of 0.7 to 2.3 µm and time-frame intervals of 0.07 to 0.21s. Our experiments were 346 conducted on HEp-2 or A549 cells which are very thin as compared to some other cell types. Expanding 347 the method to other cells is likely to require further optimization. In these cells, these parameters 348 allowed a proper automatic tracking of mobile RNPs that are visible in the analyzed volume on several 349 successive images. The potential of our method allowed us to generate high-throughput live imaging 350 datasets otherwise inaccessible with more classical approaches. We were able to track hundreds of 351 particles per cell and provide a robust characterization of vRNPs movements in different conditions. A 352 large proportion of the observed vRNPs appeared immobile or animated by low amplitude "Brownian 353 movements". The resolution of our microscopy methods (around 250nm) does not allow fine 354 characterization of very low amplitude non-directional movements. In constrat, during the 60s 355 acquisitions we performed, we observed that about 10% of the vRNPs were animated by fast and 356 directional movements." These are believed to be the ones actively trafficking. The characteristics of 357 these motions, in particular the maximum speed above 1-2 µm/s are in themselves suggestive of MT-358 related transport (32). 359 The importance of the microtubule network in RSV multiplication has already been established by 360 previous studies. Treatment of infected cells with nocodazole was shown to reduce virion production 361 by about 1 log with no indication of which stage of multiplication was disrupted (22,33). Here, our 362 results clearly establish that vRNPs are transported rapidly and over long distances on the MT network. 363 Indeed, we observed vRNPs moving on MTs and were able to verify the almost complete 364 disappearance of these movements upon disassembly of the MT network. Many negative RNA viruses 365 also hijack the MT network and its associated motors for the transport of their RNPs. For example, 366 RNPs from Influenza A , Sendai or measles viruses move along this network (25,26,(37)(38)(39). As what has 367 been observed for RSV, depolymerization of the MT network does not completely abolish the 368 replication of these viruses (26,40,41). For influenza viruses, conflicting results were reported on the 369 effect of nocodazole treatment on viral multiplication (42)  data support the following model for RSV RNPs export: 1) vRNPs transiently associate with Rab11a-389 positive vesicles 2) these Rab11a-positive vesicles transport vRNPs rapidly and over long distances 390 along the microtubule network. Moreover, RSV not only diverts the Rab11 pathway for its RNPs 391 transport but also manipulates it. Indeed, we observed that RSV infection increases transferrin 392 recycling, consistent with previous analysis of Rab11a vesicles motion that revealed increase in velocity 393 and in high distance displacement in RSV infected cells (38). 394 In recent years Rab11 has emerged as a key cellular factor involved in various stages of the 395 multiplication of several viruses. Some viruses enter the cell using the ERC as suggested by the 396 association of HHV8 or Dengue viral capsids with Rab11 endosomes soon after infection (43,44). 397 Furthermore, it has previously been shown that effectors of the Rab11 pathway: FIP1, FIP2 and 398 MyosinVb support RSV release at the apical side of the membrane. FIP2 is even thought to regulate 399 the length of viral filaments (45,46) suggesting an involvement of the ERC in the budding steps of RSV. 400 Rab11 and its partner FIP3 have also been implicated in the budding of influenza viruses (47) (26)(27)(28)(39)(40)(41)47,(49)(50)(51). However, the precise 407 mechanisms of vRNPs associations with Rab11 and the pathways involved in transport remain to be 408 defined. The numerous studies of the export of Influenza virus RNPs have shown that vRNPs 409 recruitment depends on a direct protein-protein interaction between a unit of the viral polymerase 410 complex PB2 and the active form of Rab11a, and that the Kinesin Kif13a is one of the motors involved 411 in their transport to the membrane (39,41,52). The L polymerase of Sendai virus has also been shown 412 to be involved in the recruitment of vRNPs to ERC vesicles (53). Rab11a is co-immunoprecipitated with 413 RSV L suggesting that the RSV polymerase could similarly promote Rab11a-vRNP association. However, 414 since the whole vRNP is captured in our experiments, further investigations are needed to determine 415 if L is critical for recruiting Rab11. 416 In conclusion, our results establish that RSV RNPs associated with Rab11a vesicles are transported 417 along MTs. These data are a first step forward in the understanding of vRNP export. They pave the way 418 for further work aimed at identifying the viral proteins, the Rab11 cellular partners and the molecular 419 motors involved in vRNPs transport. In the future, interactions between RSV and the ER pathway could 420 represent a new therapeutic target to block the late stages of the RSV replication cycle.

Virus and plasmids 432
All the viral sequences were derived from the human RSV strain Long, ATCC VR-26. The wild-type RSV, 433 the RSV-GFP, the RSV-GFP-N and the RSV-L-GFP were rescued by reverse genetics as previously 434 described (30,54). To construct the reverse genetic vector for RSV-GFP-N, the GFP-N coding sequence 435 was amplified from the pmGFP-N vector (11) by PCR using specific primers (sequence available upon 436 request) containing RSV gene start and gene end sequences and was cloned into a Eco105I restriction 437 site between the RSV M and SH genes in the pACNR-rHRSV (30). The nucleotide sequence of RSV-GFP-438 N was deposited in the Genbank nucleotide database with accession code OM326756. pmCherry-439 Rab11a was constructed by inserting the Cherry coding sequence in peGFP-Rab11a (55) (kind gift from 440 Dr Sauvonnet) between NheI and XhoI restriction sites. All constructs were verified by sequencing. 441 Experiments were performed with viral stock amplified on HEp-2 cells at 37 °C after three to five 442 passages. Plaque assay were performed at 37 °C on HEp-2 cells using Avicel overlay as previously 443 described (30). 444

Antibodies and reagents 445
The rabbit polyclonal anti-P and anti-N were obtained by repeated injection of purified recombinant 446 protein produced in Escherichia coli as previously described (56). The rabbit anti-Rab11a is from 447 Thermofisher, the rabbit anti-EEA1 and rabbit anti-HA from Cell signaling, the rat anti-HA is from 448 Roche, and the mouse anti-F was from Abcam. The mouse monoclonal anti-P 021/2P (57) is a kind gift 449 from Dr JF Eléouët. Secondary antibodies raised against mouse or rabbit IgG (H+L) and conjugated to 450 Alexa Fluor 488, 594 or 647 were from Thermofisher. Secondary antibodies raised against mouse or 451 rabbit IgG (H+L) and conjugated to horseradish peroxidase were from Promega. DMSO, Nocodazole 452 and Cytochalasin D were from Sigma. Live staining of Microtubules was achieved with SiR-tubulin Kit 453 647 as described by the manufacturer (Spirochrome). 454

PAGE. 495
Live Imaging 496 Live imaging was performed on A549 or HEp-2 cells seeded on Ibidi µ-Slide Angiogenesis, infected and 497 treated as described above. One-minute time-lapse acquisitions were performed at 37°C and 5% CO2 498 using an inverted confocal microscope with a 100x oil-immersion lens (Plan-APOCHROMAT), a CSU-499 X1 spinning-disk head (Yokogawa, Japan) and a sCMOSPRIME 95B (Photometrics) camera. The whole 500 setup was driven with MetaMorph software (Molecular Devices, Sunnyvale, CA, USA). One to three z 501 sections with a step of 0.8 µm were acquired at 70 to 210 ms intervals for 1 min. Laser intensity was 502 set between 10-20% power, and acquisition time was 50 ms. The raw data were processed using ImageJ 503 software (National Institutes of Health, Bethesda, MD, USA) to perform maximum projections of z 504 stacks images and background subtraction. Tracks analysis was performed on Imaris software 9.5.0 505 (Bitplane Inc.). Briefly fluorescent spots were identified using Imaris built-in spot generation algorithm 506 (with a seeding spot size of 250 nm). Spot generation was achieved manually for each dataset using 507 intensity at the center of each spot as the threshold. Quality threshold was fixed above 20.0. The spots 508 were tracked over time to generate motion statistics for each cell using autoregressive motion 509 algorithm (max distance 1µm, max gap size 2). Imaris output file "Position.csv" was further used to 510 calculate the characteristic of each tracks (track duration, track length, track displacement, track 511 maximum speed, mean track velocity) and to filter tracks using dedicated PYTHON Script available at 512 GitHub (https://github.com/mawelti/RSV-RNP-TrackAnalysis). All tracks smaller than 4 steps were 513 filtered. Coordinates of the IBs area were entered manually to remove false tracks generated by 514 frequent false detections due to high background signal in the vicinity of the IBs (see Fig S2a,b). Tracks 515 whose instant speed is always lower than 50% of the maximum instant speed were also filtered to 516 remove false link between 2 slow moving objects (See Fig S2c, d, e). Smoothed instant speed were 517 calculated as the ratio between the minimum distance between the first and the fourth of 4 518 consecutive positions and the time interval between these positions. We choose to smooth the instant 519 speed because time interval between 2 frames being very small (0.07 to 0.21s), displacement of 250nm 520 corresponding to position error of diffracted limited microscopy resulted in high instant speed (1.2 to 521 3.6 µm/s) (Fig S2 f). To compare the effect of treatments, statistical comparisons between median were 522 performed in Prism software (Graphpad Inc.) as indicated in legends. All tests used in this report are 523 two-sided. For visualization, the raw data were processed using ImageJ software (National Institutes  524 of Health, Bethesda, MD, USA). Images stacks were processed as maximum projections and visualized 525 after gaussian filter fixed at 0.5. 526 Acknowledgements: We thank Marie Galloux, Jean-Francois Eléouët, Nadia Naffakh and Cédric 527 Delevoye for sharing protocols and reagents, and for helpful discussions. We are grateful to Nathalie 528 Sauvonnet for Rab11 plasmids and helpful discussions. We thank Jennifer Risso-Ballester, Cédric Diot 529 and Sabine Blouquit-Laye for insightful discussions and critical reading of the manuscript. We thank 530 Aude Jobart-Malfait and Cymages platform for access to the Leica SP8 microscope and Olympus 531 FV3000 inverted confocal microscope, which was supported by grants from the region Ile-de-France. 532 We also acknowledge the ImagoSeine core facility of the Institut Jacques Monod and thank Xavier 533 Baudin for support for spinning disk imaging. We acknowledge support from INSERM and Versailles 534 Saint-Quentin University. This work was supported by ATIP-AVENIR INSERM program and the 535 Fondation Del Duca -Institut de France. 536 537